There is a fundamental gap in understanding of how the misfolding of tau relates to the enormous phenotypic diversity observed in human tauopathies. Persistence of this gap constitutes an important problem because, until it is filled, a molecular understanding of the processes that determine disease progression will remain largely illusive. The long-term goal is to understand the pathways of tau misfolding and the processes of interneuronal spreading. The objective of this proposal is to use newly developed seeding methodology in conjunction with double electron-electron resonance (DEER) spectroscopy (a method that measures the distances between two paramagnetic reporter molecules) to determine the conformations of polypeptide chains in tau filaments. The central hypothesis is that conformationally distinct tau filaments are associated with different disease phenotypes. This hypothesis has been formulated based on preliminary data produced in the applicant's lab that reveal a robust seeding barrier between distinct isoforms of tau and structural polymorphism of filaments composed of identical isoforms. The rationale for this project is that once differences in filament structure are known, the effects of distinct conformers on cell transfer mechanisms can be tested, and the inhibition of these processes can be investigated. Supported by strong preliminary data the central hypothesis will be tested by pursuit of the following three specific aims. 1) Determine the seeding properties of tau filaments. A novel acrylodan-based fluorescence assay will be used to investigate the seeding properties of synthetically and brain-derived tau filaments and to characterize a newly discovered seeding barrier between tau isoforms. 2) Identify conformational differences between tau filaments. DEER spectroscopy and seeded filament growth will be used to determine conformational differences between tau filaments at the molecular level. 3) Determine filament properties of phosphorylated tau and tau disease mutants. The effects of phosphorylations and disease-related mutations on filament conformation will be investigated. Changes in seeding barriers between tau isoforms will serve to identify structural changes. Preliminary data have identified a mutation that causes a switch in seeding properties. Filaments of this mutant will be analyzed in detail using distance measurements between paramagnetic reporter groups. The research is innovative, because it uses a new, sensitive approach to resolve structural differences between filaments, namely conformational templating combined with double electron-electron resonance spectroscopy. The proposed research is significant, because the results will provide a first general model of filament diversity and its relationship to human tauopathies. Such knowledge has the potential to lead to new therapeutic strategies, including the production of novel monoclonal antibodies and small molecule inhibitors that selectively interfere with specific propagation routes, slowing or reversing the progression of tau-mediated diseases.